[1]谭铭辉,梁宇灵,黄文斌,等.Galectin-3诱导大鼠骨髓间充质干细胞分化为肝样细胞[J].南方医科大学学报,2018,(09):1076.[doi:10.12122/j.issn.1673-4254.2018.09.09]
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Galectin-3诱导大鼠骨髓间充质干细胞分化为肝样细胞()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2018年09期
页码:
1076
栏目:
出版日期:
2018-09-30

文章信息/Info

Title:
Galectin-3 induces differentiation of rat bone marrow mesenchymal stem cells into hepatocyte-like cells
作者:
谭铭辉梁宇灵黄文斌程远蒋泽生何国林高毅潘明新
关键词:
半乳糖凝集素-3骨髓间充质干细胞肝样细胞诱导分化基因芯片信号通路Hippo通路
Keywords:
galectin-3 bone marrow mesenchymal stem cells hepatocyte-like cells differentiation signaling pathways Hippo signaling pathway
DOI:
10.12122/j.issn.1673-4254.2018.09.09
摘要:
目的探索应用Galectin-3诱导大鼠骨髓间充质干细胞分化为肝样细胞的效果,筛选出所涉及的信号通路,并验证Hippo 信号通路。方法对大鼠股骨骨髓间充质干细胞进行提取、分离、传代培养及鉴定。取第3代细胞进行诱导培养,分为SD大鼠 BMSCs(A组)、SD大鼠BMSCs+0.5 μg/mL Gal-3(B组)、SD大鼠BMSCs+20 ng/mL HGF(C组)、SD大鼠BMSCs+0.5 μg/mL Gal-3+20 ng/mL HGF(D组)和大鼠肝细胞(E组)。分别在诱导后7、14、21、28 d进行细胞形态学观察、糖原染色观察,并行实时 定量PCR 以及Western blot 检测,鉴定其分化情况。取B组细胞行基因芯片检测。将BMSCs 与Gal-3、Gal-3+XMU-MP-1 (Hippo信号通路抑制剂)分组进行诱导,Western Blot检测YAP、P-YAP、ALB、AFP、CK-18。结果大鼠股骨骨髓分离出的细胞 符合骨髓间充质干细胞特性。诱导后,B、C、D组BMSCs形态逐渐向肝样细胞转化,D组28 d时细胞形态与肝细胞相对比相似 度最高。糖原染色28 d 时A组无染色,B、C、D组染色较前增多,B、C组间无明显差异(P>0.05),D组染色率最高(P<0.05)。 Q-PCR检测AFP、ALB、CK-18表达逐渐升高,28 d时B组与C组各指标较接近(P<0.05)。Gal-3与HGF对AFP、ALB的mRNA 表达不存在交互效应(AFP:F=0.236, P=0.640;ALB:F=50.639, P=0.000),对CK-18 的mRNA表达有协同效应(F=50.639, P= 0.000)。Western blot检测A组几乎不表达AFP、ALB及CK18,B、C、D组AFP、ALB及CK18蛋白表达随时间延长,逐渐增加。 基因芯片检测发现上调基因27个,下调基因62个。涉及TGF-β、PI3K-Akt及Hippo等信号通路。Gal-3及Gal-3+XMU-MP-1诱 导组的YAP表达较空白对照组增加,Gal-3+XMU-MP-1较单独应用Gal-3诱导BMSCs分化的效率高。结论Galectin-3能够单 独诱导骨髓间充质干细胞向肝样细胞分化,而且联合HGF诱导BMSCs分化的效率更高。诱导过程与TGF-β、PI3K-Akt 及 Hippo等信号通路相关。抑制Hippo信号通路可提高诱导效率。
Abstract:
Objective To investigate the effect of galectin-3 in inducing the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocyte- like cells and explore the involvement of the signaling pathways in the induced cell differentiation. Methods The third passage of cultured rat femoral BMSCs were treated with 0.5 μg/mL galectin-3, 20 ng/mL hepatocyte growth factor (HGF) or both to induce their differentiation, with untreated rat BMSCs and hepatocytes as controls. At 7, 14, 21 and 28 days of induction, the cells were examined for morphological changes followed by glycogen staining, quantitative real-time PCR and Western blotting. Gene microarray technique was used to examine the mRNA expression profile of the BMSCs induced with galectin-3. The BMSCs were also induced with galectin-3 in combination with XMU-MP-1, a Hippo signaling pathway inhibitor, after which Western blotting was performed to detect the expressions of YAP, P-YAP, ALB, AFP and CK-18 in the cells. Results The cells isolated from the femoral bone marrow of SD rats showed a consistent surface marker phenotype with the BMSCs. Induction with galectin-3, HGF, or both all resulted in gradual morphological changes of the BMSCs into hepatocyte-like cells, and the cells with a combined induction for 28 days showed the highest morphological similarity with hepatocytes. The cells induced with galectin-3, HGF, or their combination for 28 days all showed increased positivity rate of glycogen staining, which was the highest in the cells with combined induction (P<0.05) without significant difference between the cells induced with galectin-3 and HGF alone (P>0.05). Induction with galectin-3 and HGF alone both increased the expressions of AFP, ALB and CK-18 mRNAs in the cells, and their expression levels were similar between the cells at 28 days (P>0.05). Galectin-3 and HGF did not show an interactive effect on the mRNA expressions of AFP (F=0.236, P=0.640) or ALB (F=50.639, P=0.000), but had a synergistic effect on CK-18 mRNA expression (F=50.639, P=0.000). The protein expressions of AFP, ALB and CK18 were also increased in the induced cells but not detected in the cells without induction. Gene microarray results revealed 27 up-regulated genes and 62 down-regulated genes in galectin-3-induced BMSCs involving TGF-β, PI3K-Akt and Hippo signal pathways. Induction with galectin-3 and galectin-3+XMU-MP-1 increased YAP expression in the cells, and galectin-3+XMU-MP-1 was more efficient to induce the differentiation of the BMSCs. Conclusion Galectin-3 can induce the differentiation of rat BMSCs into hepatocyte-like cells, and the combination with HGF increases the efficiency of induced differentiation of the cells. TGF-β, PI3K-Akt and Hippo pathways are involved in the induced differentiation of the BMSCs, and inhibiting Hippo pathway can improve the induction efficiency.

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更新日期/Last Update: 1900-01-01